1a.Objectives (from AD-416):
Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence.

1b.Approach (from AD-416):
Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques.

3.Progress Report:

The research group at Northern Illinois University is investigating genetic regulatory mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including aflatoxin-producing Aspergillus (A.) flavus. Our major interest is the study the global fungal regulatory gene VeA (or velvet) and connected regulatory networks. This regulator is unique to fungi and it is conserved in other fungal species, particularly in the group called Ascomycetes. VeA has high potential to be used to control plant diseases caused by Aspergillus and other fungi. We further characterized the initial findings related to the connection between veA and starch degradation by A. flavus. It is known that starch degrading enzymes are necessary for successful infection of plant tissues in corn. Through high performance liquid chromatography analysis we observed that amylase activity (type of enzyme activity) is significantly reduced in the veA mutant compared to the control strain in starch medium and also in corn medium. Furthermore, preliminary experiments indicated that A. flavus mutants lacking LaeA, a gene that produces a protein described in A. nidulans to interact with VeA protein in nuclei, also presented a decrease in amylase activity compared to the control strain. In addition, we have also detected a reduction of protease activity, another enzyme possibly involved in successful fungal invasion, in the veA mutant compared to A. flavus wild-type levels. Culture supernatants of the veA deletion mutant and control strain growing on corn medium were analyzed for differences in proteins secreted into the growth medium. An initial set of data has been obtained and proteins putatively associated with hydrolytic activity in A. flavus have been identified. We have also investigated the effects of oxidative stress (type of stress involving reactive oxygen) in A. flavus and the role that VeA plays in the response to this stress. Our study revealed a reduction in survivability in the veA mutant as the concentration of the oxygen species increased in the medium, compared to the wild type strain. This result indicates that the presence of veA is necessary for a normal adaptative response under exposure to oxidative stress. Furthermore, ribonucleic acid analysis of veA mutant and wild type strain under oxidative stress indicated alterations in expression profiles of several genes involved in the HogA oxidative response signaling pathway (a type of network of proteins involved in specific effects in a cell) in the absence of veA. Interestingly, preliminary results indicated protein-protein interaction between VeA and some proteins participating in this signaling pathway. The goal of performing in silico analysis (perfomed via computer simulation) of A. flavus secondary metabolism gene clusters #27 and #39 has also been accomplished. This analysis included structural genomics and comparative genomics using genomic (entirety of an organism hereditary information) databases from other fungal species. Furthermore, collaborative efforts yielded the identification of the compound associated with cluster #39, partially conserved in other Aspergillus species. Also importantly the dark pigmented compound found in mature sclerotia (over wintering bodies or survival structures of fungi) produced by gene products of cluster #27 appears to contribute to A. flavus survival under dry conditions. Also during this reporting period the study of A. flavus NsdD and NsdC genes and their role on morphogenesis (biological process involving development) and aflatoxin production has been finalized and was resubmitted after a positive review to Eukaryotic Cell.